Layered non-fouling, antimicrobial antithrombogenic coatings

ABSTRACT

Substrates, optionally coated with an undercoating, having grafted thereto one or more non-fouling materials are described herein. The non-fouling, polymeric material can be grafted to a variety of functionalized substrate materials, particularly polymeric substrates and/or polymeric undercoatings immobilized on a substrate. The compositions described herein are highly resistant protein absorption, particularly in complex media and retain a high degree of non-fouling activity over long periods of time. The compositions described herein may also demonstrate antimicrobial and/or anti-thrombogenic activity. The non-fouling material can be grafted to a functionalized substrate, or optionally from an undercoating on the substrate, preferably without significantly affecting the mechanical and/or physical properties of the substrate material.

CROSS-REFERENCE TO RELATED APPLICATIONS

This applications claimed priority to U.S. Ser. No. 61/120,285 entitled“Synthetic Anticoagulant and Antithrombogenic Polymers” by Zheng Zhang,William Shannan O'Shaughnessy, Michael Hencke, Trevor Squier, andChristopher Loose, filed Dec. 5, 2008; U.S. Ser. No. 61/120,292 entitled“Presentation of Immobilized Molecules” by William ShannanO'Shaughnessy, Victoria E. Wagner Sinha, Zheng Zhang, Michael Hencke,Trevor Squier, and Christopher Loose, filed Dec. 5, 2008; U.S. Ser. No.61/120,302 entitled “Layered Antimicrobial and AntithrombogenicCoatings” by Zheng Zhang, William Shannan O'Shaughnessy, Michael Hencke,Trevor Squier, Michael Bouchard, and Christopher Loose, filed Dec. 5,2008; and U.S. Ser. No. 61/255,264 entitled “Layered Antimicrobial andAntithrombogenic Coatings” by Zheng Zhang, William ShannanO'Shaughnessy, Michael Hencke, Trevor Squier, Michael Bouchard, andChristopher Loose, filed Oct. 27, 2009.

FIELD OF THE INVENTION

The present invention is in the field of immobilized non-foulingcoatings, specifically coatings that resist the adhesion of biologicalmaterial and are attached to a substrate surface through a graft tomethod.

BACKGROUND OF THE INVENTION

Many different materials have been investigated to resist non-specificprotein adsorption. Chemistries utilized for this purpose includepolyethers (e.g., polyethylene glycol in particular), polysaccharidessuch as dextran, hydrophilic polymers such as polyvinylpyrrolidone orhydroxyethyl-methacrylate, heparin, intramolecular zwitterions or mixedcharge materials, and hydrogen bond accepting groups such as thosedescribed in U.S. Pat. No. 7,276,286. The ability of these materials inpreventing protein adsorption varies greatly between the chemistries. Ofthese materials, only a few resist fouling to the degree required forshort-term in vivo application. However, the few materials appropriatefor short-term application, when used for longer periods of time incomplex media or in vivo, exhibit significant fouling or materialdegradation, making them unsuitable for long-term applications.

Traditional application of biocompatible coatings, especially thoseapplied to medical device substrates, is performed by dip coating thesubstrate in a single polymer solution. For hydrophilic polymers appliedto hydrophobic substrates, this approach presents many challenges as itcan be difficult to form stable coatings. In an attempt to improvestability, hydrophilic materials have been cross-linked or copolymerizedwith hydrophobic groups. However, such approaches can have significantnegative effects on the overall coating performance, especially whenresistance to protein adsorption is desired.

There exists a need for coating formulations that overcome thelimitations described above.

Therefore, it is an object of the invention to provide non-foulingcoating formulations where an undercoating functionalized substrate isused to promote adhesion of a top coating onto a substrate surface andmethods of making and using thereof.

SUMMARY OF THE INVENTION

Functionalized substrates, optionally coated with one more undercoats,having grafted thereto one or more non-fouling polymeric materials aredescribed herein. Non-fouling coatings with varying tether chemistry orpolymer backbone chemistry provide an alternative approach to developinghighly efficient, biocompatible, and bioresponsive non-fouling coatings.In a preferred embodiment, the coatings are non-leaching. Conventionalfouling resistant or non-fouling materials and surface coatings aresusceptible to fouling over prolonged exposure to complex media or invivo environments. The materials used for many non-fouling and foulingresistant coatings, or the tethers used to immobilize the coatings on asubstrate, do not posses the stability required to coat the substratefor extended periods of time, for example, at least 7, 14, 30, 60, 90,120, 365, or 1000 days

The top coating can be immobilized directly to a functionalizedsubstrate, optionally containing an undercoating, using dispersive,covalent, coordinate, ionic or chelation type bonding. Alternatively,the top coating can be immobilized using small molecule tethers, whichare covalently or non-covalently attached to the undercoating. Thebonding between the top coating and the small molecule tethers can bedispersive, covalent, coordinate, ionic or chelation type bonding. Inanother embodiment the top coating is immobilized directly on thesubstrate surface through dispersion, covalent, coordinate, ionic, orchelation type bonding. The top coating or top coating set can bereacted with an undercoating or undercoating set before being applied orimmobilized to the substrate surface. Alternatively, the undercoatingcan be immobilized on the substrate surface followed by immobilizationof the top coating on the undercoating.

In one embodiment, a composition containing an undercoating, immobilizedon a substrate or on top of one or more preceding coatings on thesubstrate, through covalent, coordinate, dispersive, or chelation typebonding is coated with a top coating that is attached to theundercoating through covalent, coordinate, dispersive, or chelation typebonding. By functionalizing the substrate with an undercoating to aid inthe attachment of the non-fouling topcoat, superior non-foulingperformance may be achieved. For example, having an undercoatingpresenting reactive functional groups can allow for a minority ofcomplementary reactive functional groups in the topcoat to stably bindthe topcoat. The majority of the groups in the topcoat can benon-fouling groups. Having a high fraction of non-fouling groups in thetopcoat may improve non-fouling performance. Optionally, this highnon-fouling performance is achieved with stability achieved through thecovalent binding of the topcoat to the undercoat or functionalizedsubstrate.

In other embodiments, the non-fouling polymeric materials describedherein can be applied as a top coating over an undercoating such thatthe non-fouling coating degrades or does not degrade, revealing orprotecting, respectively, the coating or coatings below the top coating.Many existing non-fouling materials or tethers also contain labilefunctional groups that react in vivo resulting in arbitrary degradationof the material or release of the coating from the substrate, exposingthe underlying surface. An exposed substrate is more likely to foul and,in the case of blood contacting devices, could result in thrombusformation. In contrast to the methods previously relied upon forcreating fouling resistant and non-fouling coatings, the polymer andtether chemistries described herein not only allow for the creation ofstable non-fouling surface coatings, but also allow for tailoring thereactivity of the coatings to respond to changes in a given environment(e.g anti-inflammatory drug release, during an oxidative burst, at asite of injury).

In other embodiments, linkers used to immobilize the coating(s) areresponsive to the surrounding environment; for example, the coating isreleased or releases an encapsulated bioactive agent only under specificenvironmental changes, such as in the case of oxidative conditions, lowpH, high pH, temperature changes, exposure to radiation, including, butnot limited to microwave radiation, ultraviolet radiation, or X-rayradiation, or at a site of interest and does not release coating oragent at any other time.

In alternative embodiments, the responsive linker can re-capture orre-immobilize the coating or bioactive agent, which may be present inthe surrounding solution or retained within the device. The linkingsystem that responds to changes in physiological conditions can be usedto attach the top coating to the undercoating; to attach theundercoating to the substrate surface; and/or to attach the undercoatingto the substrate and the top coating to the undercoating.

In another embodiment the undercoating(s) are immobilized throughpolymer chain entanglements with the substrate surface and the topcoating is attached to undercoating through covalent, coordinate,dispersive, or chelation type bonding.

In other embodiments, the undercoating is incorporated into the bulksubstrate material, and the top coating is attached to the undercoatingthrough covalent, coordinate, dispersive, ionic, or chelation typebonding. The top coating itself can be immobilized through chainentanglements directly with the substrate surface.

In one embodiment, the top coating contains a polymer containing atethering segment where the tethering segment contains one or morereactive groups that can participate in covalent, coordinate,dispersive, ionic or chelation type bonding with the undercoating. In aparticular embodiment, the top coat contains the tethering segmentpolyglycidyl methacrylate, and a non-fouling segment, for example, azwitterionic polymeric material, such as polysulfobetaine,polycarboxybetaine or combinations thereof. In a preferred embodiment,the undercoat is a copolymer of glycidyl methacrylate methacrylate(GMA), 2-hydroxypropyl methacrylate (HPMA), lauryl methacrylate (LMA),and trimethoxysilyl methacrylate (TMOSMA) and the topcoat is a copolymerof carboxybetaine methacrylate (CBMA) and 2-aminoethyl methacrylate(AEMA) or a copolymer of sulfobetaine methacrylate (SBMA), glycidylmethacrylate methacrylate, and 2-hydroxypropyl methacrylate. In anotherpreferred embodiment, the undercoating is a copolymer of 2-aminoethylmethacrylate (AEMA), 2-hydroxypropyl methacrylate (HPMA), laurylmethacrylate (LMA), and trimethoxysilyl methacrylate (TMOSMA) and thetopcoat is a copolymer of carboxybetaine or sulfobetaine methacrylateand glycidyl methacrylate methacrylate.

The undercoatings and/or top coatings can be applied using a combinationof chemistries known in the art including, but not limited to,aminolysis of the substrate which exposes reactive amines available forcovalent or non-covalent bonding, click chemistry methods wherein thesurface for attachment contains azide or terminal alkyne functionalityand the coating to be immobilized contains either azide or terminalalkyne functionality wherein the surface for attachment does not containthe same functionality as the coating to be immobilized, andimmobilization through thiol reactions involving olefins,alph,beta-unsaturated carbonyls, or other thiols as in the case ofdisulfide bonding. Other chemistries can include anionic or cationicreactions, attachment of a nucleophile to an electrophile or attachmentof an electrophile to nucleophile, and ring opening methods as in thecase of an epoxide or aziridine, metathesis reactions. Organometallicreactions include chelation type bonding between a mono- ormulti-dentate organic ligand and an inorganic atom with empty d-orbitalsavailable for bonding. In some embodiments the chemistries used toimmobilize a coating or coating set can be catalyzed or un-catalyzed.

Using assays discussed herein, coating formulations can be optimized tomaximize anti-thrombotic, antimicrobial, and/or anti-adherent propertiesof substrate materials, such as materials used to prepare catheters. Forexample, for topcoats, the ratio of CBMA to AEMA monomers can be variedfrom 1:1 to 20:1 to provide maximum protein resistance while stillensuring stable immobilization to the undercoat. NMR analysis (bothproton and carbon) can be used to determine the ratio of monomer unitsincorporated into the polymer. The effect of top coat average molecularweight from 5K to 500K can be evaluated using dialysis and precipitationof top coat formulations. Effects of molecular weight distribution canbe examined using varying free radical initiation schemes includinguncontrolled initiation (which will provide a polydispersity>1.5) andhighly controlled initiation through atom transferred radicalpolymerization (which typically provide a polydispersity<1.1). Gaspermeation chromatography (GPC) with refractive index (RI) can be usedto measure the molecular weight distribution of coatings materials.

The compositions described herein resist preferably greater than 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 99.9% of theadsorption of protein from solution, for example phosphate bufferedsaline (PBS) containing protein, media, serum, or in vivo relative to anuncoated control for 1 day, 7 days, 14, 21, 30, 45, 60, 90, 120, 180,365, or 1000 days.

The compositions described herein are stable over extended periods oftime, retaining greater than 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of their non-fouling,anti-thrombotic, and/or antimicrobial properties for extended periods oftime, for example, at least 1, 7, 14, 21, 30, 45, 60, 90, 120, 180, 365,or 1000 days.

The non-fouling material can be grafted to the undercoating withoutsignificantly affecting the mechanical and/or physical properties of thesubstrate material. In one embodiment, the tensile strength, modulus,device dimensions, or combinations thereof of the coated substrate arewithin 20%, preferably within 10%, more preferably within 5%, mostpreferably within 1% of the tensile strength, modulus, devicedimensions, or combinations thereof of the uncoated substrate.

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

“Zwitterion” or “zwitterionic material” refers to a macromolecule,material, or moiety possessing both cationic and anionic groups. In mostcases, these charged groups are balanced, resulting in a material withzero net charge. Zwitterionic polymers may include both polyampholytes(e.g, polymers with the charged groups on different monomer units) andpolybetaine (polymers with the anionic and cationic groups on the samemonomer unit).

“Polymer”, as used herein, includes homopolymers and copolymers.

“Co-polymer”, as used herein, refers to any polymer composed of one ormore different monomers. The copolymer may be a random copolymer orblock copolymer, such as an AB or ABA block copolymer or a graftcopolymer.

“Antimicrobial” as used herein, refers to molecules and/or compositionsthat kill (i.e., bactericidal), inhibit the growth of (i.e.,bacteristatic), and/or prevent fouling by, microorganisms includingbacteria, yeast, fungi, mycoplasma, viruses or virus infected cells,cancerous cells, and/or protozoa.

Antimicrobial activity with respect to bacteria may be quantified usinga colonization assay pre-incubation with 50% fetal bovine serum for18-20 hours at 120 RPM at 37° C., which is preferred. Followingpre-incubation, samples are placed in Staphylococcus aureus (S. aureus,ATCC 25923) which has been diluted from an overnight culture to aplanktonic concentration of 1−3×10⁵ CFU/mL in 1% tryptone soy broth(TSB). Samples are incubated with bacteria for 24-26 hrs with agitation(120 rpm) at 37° C. The concentration of TSB varies with the organismbeing used. After incubation, the samples are placed in 3 ml PBS for 5min at 240 RPM at 37° C. to remove bacteria not tightly attached. Thenaccumulated bacteria on materials are removed by sonication in a newsolution of PBS and the total number of bacterial cells quantifiedthrough dilution plating. Preferably at least a 1, 2, 3 or 4 logreduction in bacterial count occurs relative to colonization on acontrol. Similar adherence assays are known in the art for assessingplatelet, cell, or other material adhesion to the surface. A surfacethat has a lower bacterial count on it than on reference polymers may besaid to reduce microbial colonization.

“Anti-thrombogenic”, as used herein, refers to the ability of acomposition to resist thrombus formation. Anti-thrombogenic activity canbe evaluated using ex-vivo flow loop model of thrombosis. Briefly, up to10 liters of fresh blood are collected from a single animal. This bloodis heparinised to prevent coagulation, filtered to remove particulates,and autologous radio-labeled platelets are added. Within eight hoursafter blood harvesting, coated and uncoated substrates are placed in aflow loop circuit, which pumps blood from a bath over the substrate andthen back into the bath. A second internal flow loop circuit can beestablished for substrate containing a lumen by connecting the two portsof the substrate through a 2nd peristaltic pump. Blood is pumped in theouter circuit at a rate of approximately 2.5 L/min, while blood in theinner circuit is pumped at a rate of approximately 200-400 ml/min. Aftertwo hours, the substrates are removed, inspected visually for thrombusformation, and adhered platelets quantified using a Gamma counter. Forsamples not containing a lumen, only an outer circuit may be used tomeasure thrombus on the outside of the device.

“Adhesion”, as used herein, refers to the non-covalent or covalentattachment of proteins, cells, or other substances to a surface. Theamount of adhered substance may be quantified for proteins using theassay for non-fouling activity or for bacteria with the assay forantimicrobial activity or other relevant assays.

“Bioactive agent” or “active agent” or “biomolecule”, used heresynonymously, refers to any organic or inorganic therapeutic,prophylactic or diagnostic agent that actively or passively influences abiological system. For example, a bioactive agent can be an amino acid,antimicrobial peptide, immunoglobulin, an activating, signaling orsignal amplifying molecule, including, but not limited to, a proteinkinase, a cytokine, a chemokine, an interferon, tumor necrosis factor,growth factor, growth factor inhibitor, hormone, enzyme,receptor-targeting ligand, gene silencing agent, ambisense, antisense,an RNA, a living cell, cohesin, laminin, fibronectin, fibrinogen,osteocalcin, osteopontin, or osteoprotegerin. Bioactive agents can beproteins, glycoproteins, peptides, oligliopeptides, polypeptides,inorganic compounds, organometallic compounds, organic compounds or anysynthetic or natural, chemical or biological compound.

“Non-fouling”, as used herein, means that the composition reduces orprevents the amount of adhesion of proteins, including blood proteins,plasma, cells, tissue and/or microbes to the substrate relative to theamount of adhesion to a reference polymer such as polyurethane.Preferably, a device surface will be substantially non-fouling in thepresence of human blood. Preferably the amount of adhesion will bedecreased at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, or 99.9% relative to thereference polymer.

Non-fouling activity with respect to protein, also referred to as“protein resistance” may be measured using an ELISA assay. For example,the ability of a composition to prevent the adhesion of blood proteinscan be evaluated by measuring fibrinogen absorption through ELISA.Fibrinogen is a blood protein commonly used to assess the ability of anon-fouling surface to resist adsorption, given its important role inmediating platelet and other cell attachment. Briefly, samples areincubated for 90 minutes at 37° C. in 1 mg/mL fibrinogen derived fromhuman plasma, then rinsed three times with 1×PBS and transferred toclean wells. The samples are incubated for another 90 minutes at 37° C.in 10% (v/v) fetal bovine serum to block the areas unoccupied byfibrinogen. The samples are rinsed, transferred to clean wells, andincubated for 1 hour with 5.5 ug/mL horseradish peroxidase conjugatedanti-fibrinogen in 10% (v/v) fetal bovine serum. Again the samples arerinsed and transferred to clean wells with 0.1M phosphate-citrate buffercontaining 1 mg/mL chromogen of o-phenylenediamine and 0.02% (v/v)hydrogen peroxide. Incubating at 37° C. for 20 minutes produces anenzyme-induced color reaction, which is terminated by the addition of2.0N sulfuric acid. The absorbance of light intensity can then bemeasured using a microplate reader to determine the protein adsorptionrelative to controls. Preferably the amount of adhesion will bedecreased at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, or 99.9% relative to thereference polymer. For mixed protein solutions, such as whole plasma,surface plasmon resonance (SPR) or optical waveguide lightmodespectroscopy (OWLS) can be utilized to measure surface proteinadsorption without necessitating the use of individual antigens for eachprotein present in solution. Additionally, radiolabeled proteins may bequantified on the surface after adsorption from either one protein orcomplex mixtures.

“Biocompatibility” is the ability of a material to perform with anappropriate host response in a specific situation. This can be evaluatedusing International Standard ISO 10993. Biocompatible compositionsdescribed herein are preferably substantially non-toxic. “Substantiallynon-toxic”, as used herein, means a surface that is substantiallyhemocompatible and substantially non-cytotoxic.

“Substantially Non-Cytotoxic”, as used herein, refers to a compositionthat changes the metabolism, proliferation, or viability of mammaliancells that contact the surface of the composition. These may bequantified by the International Standard ISO 10993-5 which defines threemain tests to assess the cytotoxicity of materials including the extracttest, the direct contact test and the indirect contact test.

“Substantially hemocompatible”, as used herein, means that thecomposition is substantially non-hemolytic, in addition to beingnon-thrombogenic and non-immunogenic, as tested by appropriatelyselected assays for thrombosis, coagulation, and complement activationas described in ISO 10993-4.

“A substantially non-hemolytic surface”, as used herein, means that thecomposition does not lyse 50%, preferably 20%, more preferably 10%, evenmore preferably 5%, most preferably 1%, of human red blood cells whenthe following assay is applied: A stock of 10% washed pooled red bloodcells (Rockland Immunochemicals Inc, Gilbertsville, Pa.) is diluted to0.25% with a hemolysis buffer of 150 mM NaCl and 10 mM Tris at pH 7.0. A0.5 cm² antimicrobial sample is incubated with 0.75 ml of 0.25% redblood cell suspension for 1 hour at 37° C. The solid sample is removedand cells spun down at 6000 g, the supernatant removed, and the OD414measured on a spectrophotometer. Total hemolysis is defined by diluting10% of washed pooled red blood cells to 0.25% in sterile deionized (DI)water and incubating for 1 hour at 37° C., and 0% hemolysis is definedusing a suspension of 0.25% red blood cells in hemolysis buffer withouta solid sample.

“Complex media”, as used herein, refers to biological fluids orsolutions containing proteins or digests of biological materials.Examples include, but are not limited to, cation-adjusted Mueller Hintonbroth, tryptic soy broth, brain heart infusion, or any number of complexmedia, as well as any biological fluid.

“Biological fluids” are fluids produced by organisms containing proteinsand/or cells, as well as fluids and excretions from microbes. Thisincludes, but is not limited to, blood, saliva, urine, cerebrospinalfluid, tears, semen, and lymph, or any derivative thereof (e.g., serum,plasma).

“Brush” or “Polymer Brush” are used herein synonymously and refer topolymer chains that are bound to a surface generally through a singlepoint of attachment. The polymers can be end-grafted (attached via aterminal group) or attached via a side chain or a position in thepolymer chain other than a terminal position. The polymers can be linearor branched. For example, the polymer chains described herein cancontain a plurality of side chains that contain non-fouling groups. Theside chains can consist of a single non-fouling moiety or monomer and/ora non-fouling oligomer (e.g., 2-10 monomers) or polymer (e.g., >10monomers).

“Branch” and “Branched tether,” are used interchangeably and refer to apolymer structure which originates from a single polymer chain butterminates in two or more polymer chains. The polymer may be ahomopolymer or copolymer. Branched tether polymer structures may beordered or random, may be composed, in whole or in part, of anon-fouling material, and may be utilized to immobilize one or morebioactive agents. In one embodiment, the branched tether is a dendrimer.A branched tether may be immobilized directly to a substrate or to anundercoating covering a substrate.

“Degradation products” are atoms, radicals, cations, anions, ormolecules which are formed as the result of hydrolytic, oxidative,enzymatic, or other chemical processes.

“Density”, as used herein, refers to the mass of material including, butnot limited to, non-fouling materials and bioactive agents, that isimmobilized per surface area of substrate.

“Inter-polymer chain distance”, as used herein, refers to the distancebetween non-fouling polymer chains on the surface of the substrate orundercoating. Preferably, this distance is such that the non-foulingchains decrease the penetration of fouling materials into the coatingmaterial.

“Effective surface density”, as used herein, means the range ofdensities suitable to achieve an intended surface effect including, butnot limited to, antimicrobial or non-fouling activity, as definedherein.

“Hydrophilic” refers to polymers, materials, or functional groups whichhave an affinity for water. Such materials typically include one or morehydrophilic functional groups, such as hydroxyl, zwitterionic, carboxy,amino, amide, phosphate, hydrogen bond forming, and/or ether groups.

“Immobilization” or “immobilized”, as used herein, refers to a materialor bioactive agent that is covalently or non-covalently attacheddirectly or indirectly to a substrate. “Co-immobilization” refers toimmobilization of two or more agents.

“Non-degradable” as used herein, refers to material compositions that donot react significantly within a biological environment eitherhydrolytically, reductively, enzymatically or oxidatively to cleave intosmaller or simpler components.

“Stable”, as used herein, refers to materials which retain greater than25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, or 99% of their original material properties such as surfacecontact angle, non-fouling, anti-thrombogenic, and/or antimicrobialactivity for a time of 1, 7, 14, 30, 90, 365, or 1000 days in PBScontaining protein, media, serum, or in vivo.

“Substrate”, as used herein, refers to the material on which theundercoating is applied, or which is formed all or in part ofnon-fouling material, or on which the non-fouling and/or therapeutic,diagnostic, and/or prophylactic agents are immobilized.

“Coating”, as used herein, refers to any temporary, semi-permanent orpermanent layer, or layers, treating or covering a surface. The coatingmay be a chemical modification of the underlying substrate or mayinvolve the addition of new materials to the surface of the substrate.It includes any increase in thickness to the substrate or change insurface chemical composition of the substrate. A coating can be a gas,vapor, liquid, paste, semi-solid or solid. In addition, a coating can beapplied as a liquid and solidified into a solid coating.

“Undercoat” or “Undercoating,” as used herein, refers to any coating,combination of coatings, or functionalized layer covering an entiresubstrate surface or a portion thereof under an additional coating. Inone embodiment, the undercoating is used to alter the properties of oneor more subsequent coatings or layers. The undercoating may be formedfrom a polymer or copolymer. In a preferred embodiment, the undercoat isused to aid in the immobilization of a topcoat on a substrate.

“Undercoating set,” as used herein, refers to a set or group of one ormore coatings under the top coating. This group or set of coatings canbe applied together or separately covering an entire substrate surfaceor a portion thereof.

“Topcoat” or “Top coating,” as used herein, refers to any coating,combination of coatings, or functionalized layer applied on top of oneor more undercoatings, another top coating, or directly to a substratesurface. A top coating may or may not be the final coating applied to asubstrate surface. In one embodiment a top coat is covalently attachedto an undercoating. In another embodiment a top coating is encapsulatedin a protective coating, which helps extend the top coatings storagelife. In a preferred embodiment, the topcoat includes polymericmaterial.

“Top coating set,” as used herein, refers to a set or group of one ormore coatings on top of one or more undercoatings.

“Functionalized substrate”, as used herein, refers to a substrate onwhich the number of reactive or functional groups has been increasedand/or the identity of functional groups has been changed. This may beaccomplished by making chemical alterations on the surface withtechniques including, but not limited to, aminolysis. In otherembodiments, this may be accomplished by the addition of an undercoatingor undercoating set which contains functional groups.

“Non-leaching” or “Substantially non-leaching”, as used hereinsynonymously, means that the compositions retains greater than 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% of the immobilized coatingand/or bioactive agent over the course of 7, 14, 30, 90, 365, or 1000days in phosphate buffered saline (PBS), media, serum, or in vivo. Thiscan be assessed using radiolabeled active agent.

“Tether” or “tethering agent” or “Linker”, as used herein synonymously,refers to any molecule, or set of molecules, or polymer used tocovalently immobilize one or more non-fouling materials, one or morebioactive agents, or combinations thereof on a material where themolecule remains as part of the final chemical composition. The tethercan be either linear or branched with one or more sites for immobilizingbioactive agents. The tether can be any length. However, in oneembodiment, the tether is greater than 3 angstroms in length. The tethermay be non-fouling, such as a monomer, oligomer, or polymer or anon-fouling non-zwitterionic material. The tether may be immobilizeddirectly on the substrate or on a polymer, either of which may benon-fouling.

“Non-naturally occurring amino acid”, as used herein, refers to anyamino acid that is not found in nature. Non-natural amino acids includeany D-amino acids, amino acids with side chains that are not found innature, and peptidomimetics. Examples of peptidomimetics include, butare not limited to, b-peptides, g-peptides, and d-peptides; oligomershaving backbones which can adopt helical or sheet conformations, such ascompounds having backbones utilizing bipyridine segments, compoundshaving backbones utilizing solvophobic interactions, compounds havingbackbones utilizing side chain interactions, compounds having backbonesutilizing hydrogen bonding interactions, and compounds having backbonesutilizing metal coordination. * All of the amino acids in the humanbody, except glycine, exist as the D and L forms. Nearly all of theamino acids occurring in nature are the L-forms. D-forms of the aminoacids are not found in the proteins of higher organisms, but are presentin some lower forms of life, such as in the cell walls of bacteria. Theyalso are found in some antibiotics, among them, streptomycin,actinomycin, bacitracin, and tetracycline. These antibiotics can killbacterial cells by interfering with the formation of proteins necessaryfor viability and reproduction. Non-naturally occurring amino acids alsoinclude residues, which have side chains that resist non-specificprotein adsorption, which may be designed to enhance the presentation ofthe antimicrobial peptide in biological fluids, and/or polymerizableside chains, which enable the synthesis of polymer brushes using thenon-natural amino acid residues within the peptides as monomeric units.

“Polypeptide”, “peptide”, and “oligopeptide” encompasses organiccompounds composed of amino acids, whether natural, synthetic ormixtures thereof, that are linked together chemically by peptide bonds.Peptides typically contain 3 or more amino acids, preferably more than 9and less than 150, more preferably less than 100, and most preferablybetween 9 and 51 amino acids. The polypeptides can be “exogenous,” or“heterologous,” i.e. production of peptides within an organism or cellthat are not native to that organism or cell, such as human polypeptideproduced by a bacterial cell. Exogenous also refers to substances thatare not native to the cells and are added to the cells, as compared toendogenous materials, which are produced by the cells. The peptide bondinvolves a single covalent link between the carboxyl group(oxygen-bearing carbon) of one amino acid and the amino nitrogen of asecond amino acid. Small peptides with fewer than about ten constituentamino acids are typically called oligopeptides, and peptides with morethan ten amino acids are termed polypeptides. Compounds with molecularweights of more than 10,000 Daltons (50-100 amino acids) are usuallytermed proteins.

“Antimicrobial peptide” (“AmP”), as used herein, refers tooligopeptides, polypeptides, or peptidomimetics that kill (i.e., arebactericidal) or inhibit the growth of (i.e., are bacteristatic)microorganisms including bacteria, yeast, fungi, mycoplasma, viruses orvirus infected cells, and/or protozoa.

“Coupling agent”, as used herein, refers to any molecule or chemicalsubstance which activates a chemical moiety, for example on a bioactiveagent or on the material to which it will be attached, to allow forformation of a covalent or non-covalent bond between the bioactive agentand the material to which it will be attached, wherein the material doesnot remain in the final composition after attachment.

“Cysteine”, as used herein, refers to the amino acid cysteine or asynthetic analogue thereof, wherein the analogue contains a freesulfhydryl group.

“Membrane-targeting antimicrobial agent”, as used herein, refers to anyantimicrobial agent that retains its bactericidal or bacteriostaticactivity when immobilized on a substrate and can therefore be used tocreate an immobilized antimicrobial surface. In one embodiment, themembrane-targeting antimicrobial agent is an antimicrobial peptide, andin another embodiment it is a quaternary ammonium compound or polymer.“Immobilized bactericidal activity” as used herein, refers to thereduction in viable microorganisms including bacteria, yeast, fungi,mycoplasma, viruses or virus infected cells, and/or protozoa thatcontact the surface. For bacterial targets, bactericidal activity may bequantified as the reduction of viable bacteria based on the ASTM 2149assay for immobilized antimicrobials, which may be scaled down for smallsamples as follows: an overnight culture of a target bacteria in agrowth medium such as Cation Adjusted Mueller Hinton Broth, is dilutedto approximately 1×10⁵ cfu/ml in pH 7.4 Phosphate Buffered Saline usinga predetermined calibration between OD600 and cell density. A 0.5 cm²sample of immobilized antimicrobial surface is added to 0.75 ml of thebacterial suspension. The sample should be covered by the liquid andshould be incubated at 37° C. with a sufficient amount of mixing thatthe solid surface is seen to rotate through the liquid. After 1 hour ofincubation, serial dilutions of the bacterial suspension are plated onagar plates and allowed to grow overnight for quantifying the viablecell concentration. Preferably at least a 1, 2, 3 or 4 log reduction inbacterial count occurs relative to a control of bacteria in phosphatebuffered saline (PBS) without a solid sample.

The term “alkyl” refers to the radical of saturated or unsaturatedaliphatic groups, including straight-chain alkyl, alkene, and alkynegroups, branched alkyl, alkene, or alkyne groups, cycloalkyl(alicyclic),cycloalkene, and cycloalkyne groups, alkyl, alkene, or alkynesubstituted cycloalkyl, cycloalkene, or cycloalkyne groups, andcycloalkyl substituted alkyl, alkene, or alkyne groups. In preferredembodiments, a straight chain or branched chain alkyl has 30 or fewercarbon atoms in its backbone (e.g., C₁-C₃₀ for straight chain, C₃-C₃₀for branched chain), preferably 20 or fewer carbons, more preferablyless than 10 carbons atoms, most preferably less than 7 carbon atoms.Likewise, preferred cycloalkyls have from 3-10 carbon atoms in theirring structure, and more preferably have 5, 6 or 7 carbons in the ringstructure.

It will be understood that “substitution” or “substituted” includes theimplicit proviso that such substitution is in accordance with permittedvalence of the substituted atom and the substituent, and that thesubstitution results in a stable compound, e.g., which does notspontaneously undergo transformation such as by rearrangement,cyclization, elimination, etc.

As used herein, the term “substituted” is contemplated to include allpermissible substituents of organic compounds. In a broad aspect, thepermissible substituents include acyclic and cyclic, branched andunbranched, carbocyclic and heterocyclic, aromatic and nonaromaticsubstituents of organic compounds. Illustrative substituents include,but are not limited to, aryl, heteroaryl, hydroxyl, halogen, alkoxy,nitro, sulfhydryl, sulfonyl, amino (substituted and unsubstituted),acylamino, amido, alkylthio, carbonyl groups, such as esters, ketones,aldehydes, and carboxylic acids; thiolcarbonyl groups, sulfonate,sulfate, sulfinylamino, sulfamoyl, and sulfoxido.

The permissible substituents can be one or more and the same ordifferent for appropriate organic compounds. For purposes of thisinvention, the heteroatoms such as nitrogen may have hydrogensubstituents and/or any permissible substituents of organic compoundsdescribed herein which satisfy the valences of the heteroatoms. Thispolymers described herein are not intended to be limited in any mannerby the permissible substituents of organic compounds.

II. Compositions

A. Substrates

The non-fouling material may be grafted to a variety of differentundercoatings immobilized on a variety of different substrates. Theundercoating can be immobilized covalently or non-covalently on thesubstrate. Examples of suitable substrate materials include, but are notlimited to, metallic materials, ceramics, polymers, woven and non-wovenfibers, inert materials such as silicon, and combinations thereof. Inone embodiment, the substrate is a material other than gold or glass.

Suitable metallic materials include, but are not limited to, metals andalloys based on titanium, such as unalloyed titanium (ASTM F67) andtitanium alloys, such as ASTM F1108, Ti-6Al-4V ELI (ASTM F136), Nitinol(ASTM F2063), nickel titanium alloys, and thermo-memory alloy materials;stainless steel (ASTM F138 and F139), tantalum (ASTM F560), palladium,zirconium, niobium, molybdenum, nickel-chrome, or certain cobalt alloysincluding Stellite, cobalt-chromium (Vitallium, ASTM F75 and Wroughtcobalt-chromium (ASTM F90)), and cobalt-chromium-nickel alloys such asELGILOY® and PHYNOX®.

Suitable ceramic materials include, but are not limited to, oxides,carbides, or nitrides of the transition elements such as titaniumoxides, hafnium oxides, iridium oxides, chromium oxides, aluminumoxides, and zirconium oxides. Silicon based materials, such as silica,may also be used.

Suitable polymeric materials include, but are not limited to,polystyrene and substituted polystyrenes, polyalkylenes, such aspolyethylene and polypropylene, poly(urethane)s, polyacrylates andpolymethacrylates, polyacrylamides and polymethacrylamides, polyesters,polysiloxanes, polyethers, poly(orthoesters), poly(carbonates),poly(hydroxyalkanoate)s, polyfluorocarbons, PEEK, Teflon, silicones,epoxy resins, KEVLAR®, NOMEX®, DACRON®, nylon, polyalkenes, phenolicresins, PTFE, natural and synthetic elastomers, adhesives and sealants,polyolefins, polysulfones, polyacrylonitrile, biopolymers such aspolysaccharides and natural latex copolymers thereof, and combinationsthereof. In one embodiment the substrate is a medical grade polyurethaneor CARBOTHANE®, aliphatic polycarbonate-based polyurethanes, availablefrom Lubrizol Corporation, blended with appropriate extrusion agents andplasticizers, possibly one already approved by the FDA or otherappropriate regulatory agency for use in vivo.

The substrates may optionally contain a radiopaque additive, such asbarium sulfate or bismuth to aid in radiographic imaging.

Substrates may be in the form of, or form part of, films, particles(nanoparticles, microparticles, or millimeter diameter beads), fibers(wound dressings, bandages, gauze, tape, pads, sponges, including wovenand non-woven sponges and those designed specifically for dental orophthalmic surgeries), surgical, medical or dental instruments, bloodoxygenators, ventilators, pumps, drug delivery devices, tubing, wiring,electrodes, contraceptive devices, feminine hygiene products,endoscopes, grafts (including small diameter<6 mm), stents (includingcoronary, ureteral, renal, biliary, colorectal, esophageal, pulmonary,urethral, and vascular), stent grafts (including abdominal, thoracic,and peripheral vascular), pacemakers, implantablecardioverter-defibrillators, cardiac resynchronization therapy devices,cardiovascular device leads, ventricular assist devices and drivelines,heart valves, vena cava filters, endovascular coils, catheters(including central venous, peripheral central, midline, peripheral,tunneled, dialysis access, urinary, neurological, peritoneal,intra-aortic balloon pump, angioplasty balloon, diagnostic,interventional, drug delivery, etc.), catheter connectors and valves(including needleless connectors), intravenous delivery lines andmanifolds, shunts, wound drains (internal or external includingventricular, ventriculoperitoneal, and lumboperitoneal), dialysismembranes, infusion ports, cochlear implants, endotracheal tubes,tracheostomy tubes, ventilator breathing tubes and circuits, guidewires, fluid collection bags, drug delivery bags and tubing, implantablesensors (e.g., intravascular, transdermal, intracranial), ophthalmicdevices including contact lenses, orthopedic devices (including hipimplants, knee implants, shoulder implants, spinal implants (includingcervical plates systems, pedicle screw systems, interbody fusiondevices, artificial disks, and other motion preservation devices),screws, plates, rivets, rods, intramedullary nails, bone cements,artificial tendons, and other prosthetics or fracture repair devices),dental implants, periodontal implants, breast implants, penile implants,maxillofacial implants, cosmetic implants, valves, appliances,scaffolding, suturing material, needles, hernia repair meshes,tension-free vaginal tape and vaginal slings, prosthetic neurologicaldevices, tissue regeneration or cell culture devices, or other medicaldevices used within or in contact with the body or any portion of any ofthese.

In one embodiment, the substrate is a vascularly inserted catheter suchas a peripherally inserted central catheter (PICC), central venouscatheter (CVC), or hemodialysis catheter, venous valves, punctual plugs,and intra-ocular devices and implants. In another embodiment, thesubstrate is a vascularly inserted catheter formed from a medical gradepolyurthethane or CARBOTHANE® or formed from a material coated with amedical grade polyurethane or CARBOTHANE®.

The non-fouling materials can also be added to paints and other coatingsand filters to prevent mildew, bacterial contamination, and in otherapplications where it is desirable to prevent fouling, such as marineapplications (ship hull coatings), fuel tanks, oil pipelines, industrialpiping, pharmaceutical equipment, drug delivery devices such asinhalers, contact lenses, dental implants, coatings for in vivo sensors,textiles such as hospital drapes, gowns, or bedding, ventilationconduits, doorknobs, devices for separations, such as membranes formicrobial suspension, biomolecule separation, protein fractionation,cell separation, waste water treatment, water purification, bioreactors,and food processing.

These materials can also be used to treat surfaces of fibers,particulates and films for the applications of textiles, additives,electric/optical appliances, packaging materials and colorants/inks.

1. Effective Surface Area

In addition to the chemical composition of the substrate, the micro- andnano-structure of the substrate surface may be useful to maximize thesurface area available for undercoating attachment. For metallic andceramic substrates, increased surface area can be created throughsurface roughening, for example by a random process such as plasmaetching. Alternatively, the surface can be modified by controllednano-patterning using photolithography. Polymeric substrates can also beroughened as with metallic and ceramic substrates. For alternativeapplications, creating a polished or smoother surface may enhancenon-fouling properties of the material. The surface can be modified toenhance the attachment and stability of an undercoating or anundercoating set. Alternatively, the surface may be polished or smoothedto reduce surface area as this may reduce physical features which couldtrap fouling agents. Further, having a defined roughness with physicalfeatures of specified sizes and distributions may control theinteraction of bacteria, proteins, or other fouling agents with thesurface. Each of these roughness variants may be enhanced with theaddition of a non-fouling coating.

2. Surface Microstructure

In the case where a greater density of non-fouling material is desired,the creation of microstructure on the undercoating can create more areafor immobilizing the undercoating to the surface, without increasing theapparent surface area of the substrate. For polymeric substrates,including hydrogel networks, this surface morphology can be createdthrough appropriate polymer structural design.

B. Undercoatings

The substrate has immobilized thereon one or more undercoatings. Theundercoating can be immobilized covalently or non-covalently to thesubstrate surface. In some embodiment, the undercoating(s) areimmobilized on the substrate through polymer chain entanglements withthe substrate surface. Examples of non-covalent interactions include,but are not limited to, ionic bonds, coordination, dispersion,chelation, and combinations thereof. In those embodiments where theunderlayer is covalently attached to the substrate, the undercoating canbe immobilized directly on the substrate surface or through a linker ortether. The linker or tether can be part of the undercoating or can begrafted to or from the surface of the substrate prior to application ofthe undercoating.

One function of the undercoating is to provide reactive functionalgroups to immobilize the top coating. In preferred embodiments, one ormore functional groups on the undercoating and one or more complimentaryreactive functional groups in the topcoat may be used to immobilize thetopcoat on the undercoat. A range of reactive functional groups aredescribed below for both the undercoating and topcoat, though anyreactive combination may be used. In preferred embodiments, the reactivecombination includes, but is not limited to, epoxy-amine,isocyanate-carboxyl, glycidyl-anhydride, amine-anhydride,silanol-silanol, isocyanate-amine, carboxyl-amine, and hydroxyl-carboxylgroups.

Another function of the undercoating is to provide a uniform surface towhich can be attached other coatings, tethers or linkers, and/orbioactive agents. For example, medical device substrates are oftencomposed of multiple different materials, each with its own surfaceproperties. Even devices composed of a single polymer are in fact madeup of material blends and can include plasticizers, radio-opacityagents, and other additives all of which can affect substrate surfaceproperties. In order to ensure surface uniformity for maximization ofcoating adhesion and efficacy, a precoat of a single polymer may becoated on the substrate. For example, a substrate can be coated with apolymer coating, such as polyurethane, followed by immobilization of oneor more additional undercoatings and one or more top coatings on theundercoating(s). The polymer can be deposited on the substrate using avariety of techniques known in the art, such as solvent casting.

The undercoating should be mechanically stable and should not bedissolved, after curing, by the solvent used to apply the topcoat. Insome embodiments, the undercoating is applied using a process that doesno substantially impact the mechanical properties of the substrate. Thesolvent, temperature, and reaction times used during the applicationprocess may be selected to minimize the impact on the mechanicalproperties of the substrate.

The undercoating or undercoatings can be homopolymers or copolymers,such as random or block copolymers, formed by condensation or radicalpolymerization. Suitable monomers include, but are not limited to,acrylates, including substituted acrylates, such as hydroxyalkylacrylates, acrylates with primary, secondary, or tertiary amino groups,alkyl methacrylates, and reactive or crosslinkable acrylate, such asacrylates containing silyl groups, double bonds, or other reactivefunctional groups; acrylamides, including substituted acrylamides asdescribed above for acrylates; vinyl compounds; multifunctionalmolecules, such as di-, tri-, and tetraisocyanates, di-, tri-, andtetraols, di-, tri-, and tetraamines, and di-, tri-, andtetrathiocyanates; cyclic monomers, such as lactones and lactams; andcombinations thereof. Exemplary monomers are listed below:

(1) Charged methacrylates or methacrylates with primary, secondary ortertiary amine groups, such as, 3-sulfopropyl methacrylate potassiumsalt, (2-dimethylamino)ethyl methacrylate) methyl chloride quaternarysalt, [2-(methacryloyloxy)ethyl]trimethyl-ammonium chloride,methacryloyl chloride, [3-(methacryloylamino)propyl]-trimethylammoniumchloride), 2-aminoethyl methacrylate hydrochloride,2-(diethylamino)ethyl methacrylate, 2-(dimethylamino)ethyl methacrylate,2-(tert-butylamino)ethyl methacrylate, and 2-(tert-butylamino-ethylmethacrylate.(2) Alkyl methacrylates or other hydrophobic methacrylates, such asethyl methacrylate, butyl methacrylate, hexyl methacrylate, 2-ethylhexylmethacrylate, methyl methacrylate, lauryl methacrylate, isobutylmethacrylate, isodecyl methacrylate, phenyl methacrylate, decylmethacrylate, 3,3,5-trimethylcyclohexyl methacrylate, benzylmethacrylate, cyclohexyl methacrylate, stearyl methacrylate, tert-butylmethacrylate, tridecyl methacrylate, 2-naphthyl methacrylate,2,2,3,3-tetrafluoropropyl methacrylate, 1,1,1,3,3,3-hexafluoroisopropylmethacrylate, 2,2,2-trifluoroethyl methacrylate,2,2,3,3,3-pentafluoropropyl methacrylate, 2,2,3,4,4,4-hexafluorobutylmethacrylate, 2,2,3,3,4,4,4-heptafluorobutyl methacrylate,2,2,3,3,4,4,5,5-octafluoropentyl methacrylate,3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctyl methacrylate, and3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-heptadecafluorodecyl methacrylate.(3) Reactive or crosslinkable methacrylates, such as2-(trimethylsilyloxy)-ethylmethacrylate, 3-(trichlorosilyl)propylmethacrylate, 3-(trimethoxysilyl)-propyl methacrylate,3-[tris(trimethylsiloxy)silyl]propyl methacrylate, trimethylsilylmethacrylate, allyl methacrylate, vinyl methacrylate,3-(acryloyloxy)-2-hydroxypropyl methacrylate,3-(diethoxymethylsilyl)propyl methacrylate,3-(dimethylchlorosilyl)propyl methacrylate, isocyanates, such as2-isocyanatoethyl methacrylate, glycidyl methacrylate, 2-hydroxyethylmethacrylate, 3-chloro-2-hydroxypropyl methacrylate, Hydroxybutylmethacrylate, glycol methacrylate, hydroxypropyl methacrylate, and2-hydroxypropyl 2-(methacryloyloxy)ethyl phthalate.(4) Other methacrylates, such as ethylene glycol methyl ethermethacrylate, di(ethylene glycol) methyl ether methacrylate, ethyleneglycol phenyl ether methacrylate, 2-butoxyethyl methacrylate,2-ethoxyethyl methacrylate, and ethylene glycol dicyclopentenyl ethermethacrylate.

Condensation type monomers can also be used.

Acrylamide and/or methacrylamide of the monomers listed above can alsobe used, as well as other monomers with unsaturated bonds.

Multinfunctional monomers, such di, tri, or tetraacrylates orsubstituted acrylates can be used to form highly branched structureswhich can provide a higher concentration of non-fouling groups.

In one embodiment, the undercoating is a copolymer of glycidylmethacrylate (GMA), 2-hydroxypropyl methacrylate (HPMA), laurylmethacrylate (LMA), and trimethoxysilyl methacrylate (TMOSMA). Inanother embodiment, the undercoating is a copolymer of 2-aminoethylmethacrylate (AEMA), 2-hydroxypropyl methacrylate (HPMA), laurylmethacrylate (LMA), and trimethoxysilyl methacrylate (TMOSMA).

C. Top Coating

The compositions described herein contain a top coating, which isimmobilized on the outermost undercoating. The top coating can bebiodegradable or non-biodegradable, revealing or protecting,respectively, the undercoating(s) underneath the top coating.

The top coating can be immobilized covalently or non-covalently. The topcoating can be immobilized covalently to the undercoating(s) directlyvia covalent bond formation between reactive functional groups on theundercoating and the top coating. Alternatively, the top coating can beimmobilized to the undercoating(s) via a tether or linker. The tether orlinker can be immobilized on the undercoating(s) covalently ornon-covalently and the top coating can be immobilized on the linker ortether covalently or non-covalently. The top coating can also beimmobilized covalently or non-covalently on the substrate, in theabsence of an undercoating(s).

Alternatively, reactive functional groups may be created on thesubstrate directly in order to provide reactive sites to bond withreactive functional groups in the topcoat. Suitable methods for creatingreactive functional groups on the substrate are known in the art.Reactive functional groups, either on the substrate itself or on theundercoating, can be introduced, for example, by physical adsorption,chemical reaction, plasma treatment, and/or surface grafting methods.

Physical adsorption methods involve any small reactive agents which arepre-adsorbed or migrate to the surface by methods including, but notlimited to, solvent imbibing, blending, and vapor deposition.

Chemical reaction methods to create reactive functional groups include,but are not limited to, amination, hydrolysis, and silanization.

Plasma treatment methods include, but are not limited to, inert gas,reactive gas, monomers, and plasma polymerization treatment.

Surface grafting methods include, but are not limited to, surfaceinitiated reactions, such as polymerization, which include, but are notlimited to, photo-initiated, thermal-initiated, redox-initiated,controlled free radical, and anionic and cationic reactions.

In one embodiment, the top coating contains a polymer containing one ormore non-fouling segments and one or more tethering segments where thenon-fouling segment is preferably greater than 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99.0%, 99.5%, 99.9%,or 99.99% by molar ratio of the polymer.

Top coatings can be formed by synthetic means known in the artincluding, but not limited to, free radical polymerization (e.g.,thermal, UV, and/or redox), ionic polymerization, atom transfer radicalpolymerization (ATRP), nitroxide mediated polymerization (NMP),reversible addition-fragmentation polymerization (RAFT), ring openingmetathesis polymerization (ROMP), telluride mediated polymerization(TERP) or acyclic diene metathesis polymerization (ADMET).

1. Non-Fouling Materials

Surfaces which resist non-specific protein adsorption are important inthe development of biomedical materials, such as medical devices andimplants. Such coatings limit the interactions between the implants andphysiological fluids. In environments where fluids contain highconcentrations of biological proteins, such as blood contactingapplications, prevention of protein adsorption may prevent fouling ofthe device surface and/or thrombus formation. In one embodiment, thenon-fouling material is a polymeric material. Suitable polymericmaterials include, but are not limited to, zwitterionic polymers,non-zwitterionic polymers, and combinations thereof. The non-foulingpolymeric material typically a has a weight average molecular weightfrom about 1,000 Daltons to 2,000,000 Daltons, preferably from 1,000Daltons to about 1,000,000 Daltons, more preferably from about 1,000Daltons to about 500,000 Daltons, most preferably 5,000 Daltons to about500,000 Daltons.

i. Zwitterionic Materials

Zwitterions are molecules that carry formal positive and negativecharges on non-adjacent atoms within the same molecule. Both natural andsynthetic polymers, containing zwitterion functionality, have been shownto resist protein adhesion. In one embodiment, the zwitterionic monomercontains a phosphorylcholine moiety, a sulfobetaine moiety, a carboxybetaine moiety, derivatives thereof, or combinations thereof. Substratesurfaces treated with phosphorylcholine (PC), a natural zwitterionicmolecule, not only exhibit reduced protein adsorption, but also exhibitincreased blood compatibility, when compared to untreated substratesurfaces. Polymers created from phosphorylcholine are also consideredbiomimetic in addition to exhibiting the properties discussed above.

Sulfobetaine, closely resembles 2-aminoethanesulfonic acid, one of themost abundant, low molecular weight organic compounds found in animals.Sulfobetaine monomers are typically easier to handle thanphosphorylcholine and the resulting polymers are generally easier tosynthesize than the corresponding phosphorylcholine analogs.

Polycarboxybetaines are polymeric analogs of the naturally occurringzwitterion, glycine betaine. Similar to polyphosphorylcholines andpolysulfobetaines, polycarboxybetaines are another class ofzwitterionic, biomimetic polymers with exceptional resistance tobiofouling. These polymers are particularly well suited for bloodcontacting applications due to anti-thrombogenic and anticoagulantproperties unique to carboxybetaines. In addition to these properties,it is possible to design carboxybetaine monomers such that the resultingpolymers contain reactive functional groups for immobilization ofbioactive molecules. By creating carboxybetaine brushes on the surface,the dual function of resisting protein or platelet attachment and havingan actively anticoagulant group may reduce thrombosis on a surfacefurther than using either strategy alone.

Polysulfo- and polycarboxybetaines are not only biomimetic and highlyresistant to bacterial adhesion, biofilm formation, and nonspecificprotein adsorption from blood serum and plasma, they are also non-toxic,biocompatible and typically exhibit greater stability in complex mediaor in vivo when compared to both polyphosphorylcholine and poly(ethyleneglycol), which may be degraded. The application of these materials andcoatings can be further extended using biologically active agents, suchas antimicrobial peptides.

Other natural and synthetic zwitterion chemistries can be used to designnon-fouling materials for the biomedical applications described herein.Some examples of natural zwitterions chemistries that could be used fornon-fouling materials include, but are not limited to, amino acids,peptides, natural small molecules including, but not limited to,N,N,N-trimethylglycine (glycine betaine), trimethylamine oxide (TMAO),dimethylsulfoniopropionate sarcosine, lysergic acid and psilocybin.Additional synthetic zwitterions that could be used to createnon-fouling materials, include, but are not limited to, amino-carboxylicacids (carboxy betaines), amino-sulfonic acids (sulfo betaines),cocamidopropyl betaine, quinonoid based zwitterions,decaphenylferrocene, and non-natural amino acids. Natural and syntheticpolymers also include mixed charged structures with both positivecharged and negative charged moieties on the pendant groups, in the mainchains, or at the terminal groups.

Materials containing, or composed of, these natural or syntheticzwitterions, can be applied to surfaces, particularly the surfaces ofmedical devices, in order to improve biocompatibility, reducethrombogenesis (such as on the surface of stents or venous valves), andreduce fouling by proteins or bacteria present in solution. This isparticularly applicable for surfaces where non-specific binding ofproteins in solution could negatively impact the desired or necessarymechanics of a device.

In one embodiment, the non-fouling material is a zwitterionic polymergrafted from the substrate. For example, the polymer can contain one ormore monomers of Formula I:

wherein B is selected from the group consisting of:

wherein R is selected from the group consisting of hydrogen, substitutedalkyl, or unsubstituted alkyl;

E is selected from the group consisting of substituted alkyl,unsubstituted alkyl, —(CH₂)_(y)C(O)O—, and —(CH₂)_(y)C(O)NR²;

Y is an integer from 0-12;

L is absent or is a straight or branched alkyl group optionallyincluding one or more oxygen atoms;

ZI is a zwitterionic group; and

X is an integer from 3 to 1000.

In a particular embodiment, ZI is selected from the group consisting of:

wherein R₃ and R₄ are independently selected from the group consistingof hydrogen and substituted or unsubstituted alkyl;

R₅ is selected from the group consisting of substituted or unsubstitutedalkyl, phenyl, and polyether groups; and

M is an integer from 1-7.

In another embodiment, the polymer contains one or more monomers ofFormula II:

wherein B₁ and B₂ are independently selected from

R is selected from hydrogen and substituted or unsubstituted alkyl;

E is selected from substituted or unsubstituted alkylene,—(CH₂)_(p)C(O)O—, and —(CH₂)_(p)C(O)NR²—, wherein p is an integer from 0to 12,

R² is selected from hydrogen and substituted or unsubstituted alkyl;

L is a straight or branched alkylene group optionally including one ormore oxygen atoms;

-   -   P₁ is a positively charged group;    -   P₂ is a negatively charged group, such as a carboxylate group or        an SO₃ ⁻ group;    -   m is an integer from 3 to 1000; and    -   n is an integer from 3 to 1000.

In one embodiment, the positively charged group is a moiety containing aquaternary nitrogen or a cationic phosphorous group and the negativelycharged group is a moiety containing a carboxylic acid group, SO₃ ⁻, orPO₃ ⁻ group.

In still another embodiment, the polymer contains one or monomers ofFormula III, IV, or V:

wherein R is selected from and substituted or unsubstituted alkyl;

L₁ L2, and L₃ are independently a straight or branched alkylene groupoptionally including one or more oxygen atoms; and

n is an integer from 3 to 1000; and

N1 is a negatively charged group such as a carboxylate group, SO₃ ⁻group, or PO₃ ⁻ group.

In certain embodiments, an antimicrobial and/or antithromboticcomposition is provided, that contains a substrate, for example,polyurethane, covalently bound to a plurality of polymer chains. Forexample, such polymer chains may be represented by Formula I, II, III,IV, or V. In certain embodiments, the non-fouling material is a brushstructure containing one or more monomers of Formula I, II, III, V, orV. In still other embodiments, a the non-fouling material is a copolymercontaining one or more of the monomers represented by Formula I, II,III, IV, or V.

In certain embodiments, an antimicrobial and/or antithrombotic polymericcomposition is provided, that contains an undercoat covalently bound toa plurality of homopolymer chains, wherein the polymer chains optionallycontain one or more tethering segments. For example, such homopolymerchains may be represented by Formula I, II, III, or IV. In certainembodiments, the non-fouling material is a brush structure containingone or more monomers of Formula I, II, III, or IV. In still otherembodiments, a the non-fouling material is a copolymer containing one ormore of the monomers represented by Formula I, II, III, or IV.

In one embodiment, the topcoat is a copolymer of carboxybetaine and2-aminoethyl methacrylate (AEMA) or a copolymer of sulfobetainemethacrylate, glycidyl methacrylate methacrylate, and 2-hydroxypropylmethacrylate.

ii. Non-Zwitterionic Non-Fouling Materials

The topcoating can also contain a non-zwitterionic non-fouling material,alone or in combination with a zwitterionic material. These non-foulinggroups may have varying degrees of non-fouling performance in a range ofenvironments. Suitable non-zwitterionic materials include, but are notlimited to, polyethers (e.g., polyethylene glycol), polysaccharides suchas Dextran, hydrophilic polymers such as polyvinylpyrrolidone (PVP) andhydroxyethyl-methacrylate (HEMA), heparin, mixed charge materials, andmaterials containing hydrogen bond accepting groups, such as thosedescribed in U.S. Pat. No. 7,276,286. Suitable polymer structuresincluded, but are not limited to, polymers or copolymers containingmonomers of Formula I wherein ZI is replaced by a non-zwitterionic,non-fouling headgroup.

iii. Copolymers

In one embodiment, the non-fouling material is a copolymer, such as arandom copolymer or a block copolymer. Suitable non-zwitterionicmonomers include, but are not limited to, the co-monomers discussedabove with respect to the undercoating.

D. Tethers and Linkers

As discussed above, the undercoating and/or top coating can beimmobilized using a tether or linker. The stability of theundercoating(s) and/or top coating(s) may be dependent on the method ofimmobilization for each coating on the substrate surface. Variations intether chemistry can provide an opportunity to develop highly efficient,biocompatible and bioresponsive immobilized non-fouling and/or bioactiveagent coatings. The bonding between a tether molecule and the coatingand/or a bioactive agent can be covalent, non-covalent, ionic,dispersive, coordinate, chelation type bonding or combinations thereof.To ensure permanent immobilization, a non-labile or un-reactive tethercan be synthesized. Such a tether should provide a linkage that isstable in vivo between the substrate surface and the immobilizedmolecule or material.

Tethers can be formed by synthetic means known in the art including, butnot limited to, free radical polymerization, ionic polymerization, atomtransfer radical polymerization (ATRP), nitroxide mediatedpolymerization (NMP), reversible addition-fragmentation polymerization(RAFT), ring opening metathesis polymerization (ROMP), telluridemediated polymerization (TERP) or acyclic diene metathesispolymerization (ADMET). Tethers can be formed either by grafting fromthe substrate or by grafting to the substrate and subsequently graftingto the tether the non-fouling material and/or biomolecule.

In one embodiment, the linkers and tethers are responsive to thesurrounding environment. For example, the linkers and tethers mayrelease the undercoating and/or top coating under specific conditions,e.g., oxidative conditions, low pH, or when the device arrives at thedesired site. Conversely, when the composition is not in the presence ofreleasing conditions, the linker or tether re-immobilizes theundercoating and/or top coating, which may be present in the surroundingsolution or retained within a device in which the coatings are found.

Tethers and linkers may be molecules or polymers containing one or morefunctional groups including, but not limited to, divinyl compounds,diacrylates, dimethacrylates, diisocynates, diglycidyl ethers, anddimaleic anhydrides. Alternatively, hetero-bifunctional tethers may beused.

E. Fluorescent and Colormetric Labels

In one embodiment, the surface modification is stained or labeled withone or more colorimetric labels, fluorescence labels, or combinationsthereof. These labels are used to visualize the surface modificationusing the naked eye, spectroscopy, microscopy, or combinations thereof.Suitable microscopy techniques include, but are not limited to, opticalmicroscopy, fluorescent microscopy, and combinations thereof.

The surface can be stained through a chemical reaction or by physicaladsorption such as charge-charge interactions, hydrophobic interactions,or hydrophilic interactions. Labeling compounds include, but are notlimited to, compounds or derivatives of rhodamine, fluorescein,coumarin, orange B, crystal violets, toluidine blue, methyl violet,nuclear fast red, methylene blue, malachite green, magenta, acriflavine,and other azo compounds.

In another embodiment the surface modification, such as a zwitterionicpolymer, is labeled by incorporating one or more reactive labelingmonomers into the polymer backbone during polymerization. These labelingmonomers include, but not limited to, FITC-methacrylate, FITC-acrylate,rhodamine-methacrylate, rhodamine-acrylate, their derivatives or anyother fluorescent acrylate, methacrylate, acrylamide, vinyl compound,diol or diamine. Incorporation of these groups allows for convenientmeasurement of conformality and/or thickness of the coating. This may beparticularly useful as a quality control metric for conformalityverification during manufacturing of the coating on an underlyingdevice.

In another embodiment of the surface modification is stained with one ormore compounds, which could be easily visualized under an electronicmicroscope (SEM or TEM). These compounds include, but not limited toosmium tetroxide and ruthenium tetroxide.

F. Bioactive Agents

Therapeutics, diagnostic, and/or prophylactic agents can be immobilizedon a substrate. These agents can interact passively or actively with thesurrounding in vivo environment. The agents can also be used to alterthe surrounding in vivo chemistry or environment. Two or more agents canbe immobilized to a substrate surface, wherein the activity of the twoagents is greater than either of the agents alone. A substance, materialor agent that is not considered active, can become active if an activeagent is immobilized on the substance, material or agent. Active agentsinclude, but are not limited to inorganic compounds, organometalliccompounds, organic compounds or any synthetic or natural, chemical orbiological compounds of known or unknown therapeutic effect.

Cell adhesion agents can be immobilized to the compositions describedherein. The efficacy of a cell adhesion agent in binding cells incomplex environments may be enhanced by reducing non-specific proteinadsorption on the surface from which they are presented, given that cellattachment may be a competitive process with other protein adsorption.Further, there may an advantage to resisting attachment of any cellsother than those specifically targeted by the cell adhesion agent toprevent competitive blocking of the surface.

Examples of desirable cell attachment agents include, but are notlimited to, integrin binders. Exemplary integrin binders include, butare not limited to, RGD peptides, along with a number of variants thatinclude RGD motifs. Longer variants of this peptide may have morespecific target cell binding. Further, the ability to present locallydense concentrations of cell attachment agents may increase theeffectiveness of cell attachment by creating multimeric interactions.Other cell adhesion agents include, but are not limited, to REDVpeptides. Tailored integrin binders can be used for a variety ofapplications including osteointegration.

Cell adhesion agents that bind specific immune cells may also benefitfrom attachment to zwitterions. Adhesion of immune cells to thebiomaterial surface activates these cells and prefaces their phenotypicresponse, such as the transition of monocytes to macrophages that canresult, in some cases, in the fusion into undesirable foreign body giantcells. The inherent resistivity to random protein fouling thatzwitterions possess provides a unique platform to couple biomoleculesthat act as specific ligands for immune cells including neutrophils andmonocytes. Selection of appropriate ligands may prime these cells forbeneficial instead of detrimental functions. These ligands includepeptides or proteins that specifically bind immune cell receptors suchas integrins, selectins, complement, or Fc gamma. When bound to thesecell-associated proteins, such ligands may stimulate intracellularsignaling pathways that lead to responses including cytoskeletalrearrangements, production and secretion of molecules includingchemokines, cytokines and other chemoattractants, and induction ofapoptosis. Desirable behaviors that could be tailored by presentation ofbiomolecules via zwitterionic tethers may include prevention/reductionin the secretion of proinflammatory cytokines, enhancement ofphagocytosis, and modulation of the release of soluble factors thatinfluence tissue-device integration.

Osteointegration may also be promoted or induced by factors which wouldbenefit from the non-fouling properties and stable presentation ofnon-fouling materials, such as zwitterions. Osteointegration promotingagents include, but are not limited to, bone-morphogenic proteins, suchas BMP2 and shortened analogues thereof. Non-fouling surfaces, such aszwitterionic surfaces, may enhance the activity of agents designed topromote desired cell regrowth over a surface. Reducing attachment ofneutrophils and macrophages may inhibit the foreign body response andenable desired cell attachment and growth process to be favored.

Presentation of antithrombotic agents may also be more effective whentethered to non-fouling materials, such as zwitterionic materials,relative to other tethers. The process of thrombosis involves bothsurface and bulk pathways. Zwitterions have shown an ability to reduceplatelet attachment and activation, reducing one pathway. Combining anactive antithrombotic that assists in the reduction of plateletactivation or directly targets additional pathways for thrombosis with azwitterionic tether could enhance the antithrombotic effect compared toeither a non-platelet adherent surface or the antithrombotic agentalone. Suitable antithrombotic agents include, but are not limited to,thrombomodulin, heparin, reversible albumin binders, tissue plasminogenactivator binders, transglutimase, reversible NO binders, polylysine,sulphonated polymers, thrombin inhibitors including hirudin, urokinase,and streptokinase.

Device-centered infection remains a large problem. Non-foulingmaterials, such as zwitterions materials, can by themselves diminishmicrobial adhesion and retard biofilm development. Prevention ofmicrobial adhesion and biofilm can be further enhanced on non-foulingsurfaces, such as zwitterionic surfaces, by presentation ofantimicrobials including, but not limited to, membrane-targetingantimicrobial agents, antimicrobial peptides and small moleculeantimicrobial agents. Generally, antimicrobial peptides are cationicmolecules with spatially separated hydrophobic and charged regions.Exemplary antimicrobial peptides include linear peptides that form anα-helical structure in membranes or peptides that form β-sheetstructures, optionally stabilized with disulfide bridges in membranes.Representative antimicrobial peptides include, but are not limited to,cathelicidins, defensins, dermcidin, and more specifically magainin 2,protegrin, protegrin-1, melittin, 11-37, dermaseptin 01, cecropin,caerin, ovispirin, cecropin A melittin hybrid, and alamethicin, orhybrids or analogues of other AmPs. Naturally occurring antimicrobialpeptides include peptides from vertebrates and non-vertebrates,including plants, humans, fungi, microbes, and insects.

Antimicrobial peptides can be made from naturally occurring amino acids,non-naturally occurring amino acids (e.g., synthetic or semisyntheticamino acids and peptidomimetics), or combinations thereof. Antimicrobialpeptides which retain their activity when immobilized on a surface aregenerally referred to as membrane-targeting antimicrobial agents.Antimicrobial peptides can be immobilized on the non-fouling coating,the substrate, the undercoat, or combinations thereof by reacting afunctional group on the peptide with a functional group on thenon-fouling coating, the substrate, and/or the undercoat. For example,the peptide can be designed to have a cysteine residue which can be usedto immobilize the peptide on a surface by reacting the thiol group ofthe cysteine residue with a thiol-reactive group on the surface.

Tethering of these agents via non-fouling materials, such aszwitterions, should provide stable, long-term activity. Additionally,immobilization of enzymes that degrade bacterial attachment and biofilmproteins, such as glycosylases, lyases, and serine-proteases, or thosethat degrade microbial communication signal molecules, such asN-acyl-homoserine lactone acylases, could provide improved efficacy inprevention of initial microbial adhesion events and subsequent biofilmformation.

Non-fouling surfaces, such as zwitterionic surfaces, may also present aparticularly attractive surface for immobilization of biomolecules, suchas antibodies, for use as biosensors. Immobilized antibodies onnon-fouling surface surfaces, such as zwitterionic surfaces, have beendemonstrated to retain both antibody activity and antigen specificity inwhole blood. “Smart” implanted medical devices that detect undesirableactivation of specific immune pathways, such as proinflammatorycytokines, or the presence of a possible infectious agent, perhapsthrough detection of a secreted microbial toxin, could be designed, forexample, by utilizing specific antibodies or biomolecules tailored tomonitor these threats. Appropriate therapeutic strategies could then beemployed before an unfavorable outcome, such as infection, arises. Thestability of the zwitterionic molecule in vivo provides a uniqueadvantage in this type of scenario due to its longevity.

III. Methods of Making

Non-fouling surfaces, which are substantially more stable in vivo, havebeen created using graft to chemistries in combination with non-foulingpolymeric materials, such as zwitterionic polymers. The coatings createdusing these grafting methods may be more effective in their ability toretain the desired non-fouling properties over long periods in vivo,even in cases where the coating incurs slight damage (i.e.micro-scratches). In addition to long-term stability, the monomer andtether chemistries can be tailored in a manner that allows forcontrolling a coating's response to the surrounding environment as wellas controlling coating degradation if desired.

As discussed above, the undercoating(s), top coating, and/or activeagent can be immobilized covalently or non-covalently. Methods used toapply covalent coatings include, but are not limited to, dipping,spraying, blade, powder, and painting. Non-covalent methods include, butare not limited to, dipping, spraying, and painting.

Undercoatings can be formed by synthetic means known in the artincluding, but not limited to, free radical polymerization (e.g.,thermal, UV, and/or redox), ionic polymerization, atom transfer radicalpolymerization (ATRP), nitroxide mediated polymerization (NMP),reversible addition-fragmentation polymerization (RAFT), ring openingmetathesis polymerization (ROMP), telluride mediated polymerization(TERP) or acyclic diene metathesis polymerization (ADMET).

For example, the undercoating can be prepared by polymerizing one ormore monomers using polymerization methods known in the art. Thesubstrate can be coated with the undercoat by exposing the surface ofthe substrate to the undercoating material, for example, by dipping thesubstrate into a solution of the undercoat. The thickness of theundercoating can be tailored by varying the concentration of undercoatin solution and/or by increasing the number of dip steps and/or thechanging the speed of the dip step. A washing step may follow theapplication of the undercoating before the topcoat is applied.

Optionally, after the dip step, the polymer-coated substrate may becured to covalently bind the undercoating to the substrate. Followingcuring, the undercoat-coated substrate is exposed to the topcoat, forexample, by dipping the coated substrate into a solution of the topcoat.The substrate may be cured to covalently bind the topcoat to theundercoat.

Functional groups on the substrate which can be used to covalently bindthe top coat to the undercoating(s) and/or to a functionalized substrateinclude, but are not limited to, amines, which can be introduced throughaminolysis of the substrate; click chemistry methods wherein the surfacefor attachment contains azide or terminal alkyne functionality and thecoating to be immobilized contains either azide- or terminalalkyne-reactive functionality wherein the surface for attachment doesnot contain the same functionality as the coating to be immobilized; andimmobilization through thiol reactions involving olefins,alph,beta-unsaturated carbonyls, or other thiols as in the case ofdisulfide bonding.

Other chemistries can include anionic or cationic reactions,nucleophile-electrophile reactions, addition reactions, such as Michaeladdition, ring opening methods, such as epoxide or aziridine, andmetathesis reactions. Organometallic reactions include chelation typebonding between a mono- or multi-dentate organic ligands and inorganicatoms with empty d-orbitals available for bonding. In some embodimentsthe chemistries used to immobilize a coating or coating set can becatalyzed or un-catalyzed.

Coatings can be applied by simultaneously dipping the external portionin a polymer solution or dispersion to coat the external portion andflowing a polymer solution or dispersion through the intralumenalportion to coat the intralumenal portion. These unit operations are usedcommercially to modify marketed short-term antimicrobial catheters.Coating application parameters utilized to effect coating controlinclude the solvent system, percent solids and viscosity, and curetemperature and time. Suitable solvents for the undercoat include, butare not limited to, alcohols, such as methanol or ethanol. Suitablesolvents for the topcoat include, but are not limited to, water.Application and cure temperature can vary, for example between ambientand 50° C. so as not to affect physical properties of the underlyingpolyurethane substrate. Solids content can vary between 0.5-10%, withsolution viscosity no higher than 12 cP for ease of handling andapplication. Typical combined thickness of the under coat and top coatwill not exceed 100 μm; however, coating thicknesses greater than 100 μmmay be used if desired.

For example, a substrate can be coated with an undercoating by exposingthe substrate to a solution of the undercoating. In one embodiment, thesubstrate is immersed in a solution of the undercoating. The substratecan be dipped once or multiple times. The thickness of the coating canbe controlled by the number of dips and/or the rate of immersion.Following dipping, the coated substrates are typically dried to removesolvent. After drying, coated substrates can be heated, for example, 16hours at 60° C., to cure the undercoating so that it is covalently boundto the substrate.

The top coat can be applied by dipping the undercoat-coated substrateinto a solution of top coat. The thickness of the coating can becontrolled by the number of dips and/or the rate of immersion. Afterdrying, the top coat can be cured by heating the substrate, for examplefor 40 hours at 60° C.

As discussed above, many medical device substrates possess internalcavities or lumens which also necessitate coating. All disclosedapproaches can be applied both externally and internally to a medicaldevice substrate provided there is open access to the cavity. In oneembodiment the internal surface which is coated is the lumen or lumensof a catheter. If internal coating is necessary but cavity access is notavailable, medical device design may need to be altered to provide atleast temporary access tot the cavity for coating.

Bioactive agents can be immobilized onto functionalized substrates,undercoatings, and/or non-fouling materials using the chemistriesdescribed above. The chemistries can be modified as the bioactive agentand/or substrate require.

During both the chemistry and the catheter coating optimizations, coatedsubstrates can be characterized for chemical, biological, and mechanicalproperties to ensure proper alignment with key product requirements.Suitable assays include:

-   -   Attenuated Total Reflection IR (ATR-IR) can be utilized to        verify the chemical composition of the coating.    -   Scanning electron microscopy (SEM) can be utilized on the sample        cross-section to determine coating thickness. Samples are        typically flash frozen in liquid nitrogen and then freeze        fractured to prevent any distortion of the coating during        sectioning.    -   Mechanical stability of coatings can be demonstrated by        examining both activity and potential cracking (via microscopy)        after stretching and bending stresses of the catheter.    -   An enzyme-linked immunosorbent assay (ELISA) can be used to        quantify fibrinogen binding.    -   Supernatants from samples stored in PBS can be inoculated with        bacteria to confirm the lack of any leaching antimicrobial        agents that could confound biological testing results    -   A 24-hr biofilm system can be used to assess bacterial growth on        coated and control catheter segments using both S. epidermidis        and S. aureus    -   A 2-hr external flow loop with fresh bovine blood can be used to        quantify attachment of radio-labeled platelets as a measure of        thrombosis formation

Using the assays described above, coating formulations can be optimizedto maximize anti-thrombotic, antimicrobial, and anti-adherent propertiesof catheter substrate materials. For example, for topcoats, the ratio ofCBMA to AEMA monomers can be varied from 1:1 to 20:1 to provide maximumprotein resistance while still ensuring stable immobilization to theundercoat. NMR analysis (both proton and carbon) can be used todetermine the ratio of monomer units incorporated into the polymer. Theeffect of top coat average molecular weight can be evaluated usingdialysis and precipitation of top coat formulations. Effects ofmolecular weight distribution can be examined using varying free radicalinitiation schemes including uncontrolled initiation (which typicallyprovide a polydispersity>1.5) and highly controlled initiation throughatom transferred radical polymerization (which typically provide apolydispersity<1.1). Gel permeation chromatography (GPC) with refractiveindex (RI) can be used to measure the molecular weight distribution ofall coatings.

IV. Methods of Use

The materials described above may be in the form of a medical device towhich the non-fouling material is applied as a coating. Suitable devicesinclude, but are not limited to, surgical, medical or dentalinstruments, ophthalmic devices, wound treatments (bandages, sutures,cell scaffolds, bone cements, particles), appliances, implants,scaffolding, suturing material, valves, pacemaker, stents, catheters,rods, implants, fracture fixation devices, pumps, tubing, wiring,electrodes, contraceptive devices, feminine hygiene products,endoscopes, wound dressings and other devices, which come into contactwith tissue, especially human tissue.

A. Fibrous and Particulate Materials

In one embodiment, the non-fouling materials are coated directly on afibrous material, incorporated into a fibrous material or coatedindirectly on a fibrous material (e.g. coated on a different surfacecoating). These include wound dressings, bandages, gauze, tape, pads,sponges, including woven and non-woven sponges and those designedspecifically for dental or ophthalmic surgeries (See, e.g., U.S. Pat.Nos. 4,098,728; 4,211,227; 4,636,208; 5,180,375; and 6,711,879), paperor polymeric materials used as surgical drapes, disposable diapers,tapes, bandages, feminine products, sutures, and other fibrousmaterials.

Fibrous materials are also useful in cell culture and tissue engineeringdevices. Bacterial and fungal contamination is a major problem ineukaryotic cell culture and this provides a safe and effective way tominimize or eliminate contamination of the cultures, while allowingselective attachment of the desired cells through the incorporation ofdirected adhesion proteins into the material.

The non-fouling agents are also readily bound to particles, includingnanoparticles, microparticles, millimeter beads, or formed intomicelles, that have uses in a variety of applications including cellculture, as mentioned above, and drug delivery. Non-fouling,biocompatible, polymeric micelles would prevent protein denaturationpreventing activation of the immune response allowing for a morestealthy delivery of the desired therapeutic.

B. Implanted and Inserted Materials

The non-fouling material can also be applied directly to, orincorporated in, polymeric, metallic, or ceramic substrates. Suitabledevices include, but are not limited to surgical, medical or dentalinstruments, blood oxygenators, pumps, tubing, wiring, electrodes,contraceptive devices, feminine hygiene products, endoscopes, grafts,stents, pacemakers, implantable cardioverter-defibrillators, cardiacresynchronization therapy devices, ventricular assist devices, heartvalves, catheters (including vascular, urinary, neurological,peritoneal, interventional, etc.), shunts, wound drains, dialysismembranes, infusion ports, cochlear implants, endotracheal tubes, guidewires, fluid collection bags, sensors, wound treatments (dressings,bandages, sutures, cell scaffolds, bone cements, particles), ophthalmicdevices, orthopedic devices (hip implants, knee implants, spinalimplants, screws, plates, rivets, rods, intramedullary nails, bonecements, artificial tendons, and other prosthetics or fracture repairdevices), dental implants, breast implants, penile implants,maxillofacial implants, cosmetic implants, valves, appliances,scaffolding, suturing material, needles, hernia repair meshes,tension-free vaginal tape and vaginal slings, tissue regeneration orcell culture devices, or other medical devices used within or in contactwith the body or any portion of any of these.

Preferably, the non-fouling coating herein does not significantlyadversely affect the desired physical properties of the deviceincluding, but not limited to, flexibility, durability, kink resistance,abrasion resistance, thermal and electrical conductivity, tensilestrength, hardness, burst pressure, etc. In one embodiment, the tensilestrength, modulus, device dimensions, or combinations thereof of thecoated substrate are within 20%, preferably within 10%, more preferablywithin 5%, most preferably within 1% of the tensile strength, modulus,device dimensions, or combinations thereof of the uncoated substrate.

The compositions described herein resist preferably greater than 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 99.9% of theadsorption of protein from solution, for example phosphate bufferedsaline (PBS), media, serum, or in vivo relative to an uncoated controlfor 1 day, 7 days, 14, 21, 30, 45, 60, 90, 120, 180, or 365 days.

C. Non-Medical Applications

The non-fouling materials can also be added to paints and other coatingsand filters to prevent mildew, bacterial contamination, and in otherapplications where it is desirable to prevent fouling, such as marineapplications (ship hull coatings), contact lenses, dental implants,coatings for in vivo sensors, textiles such as hospital drapes, gowns,or bedding, ventilation conduits, doorknobs, devices for separations,such as membranes for microbial suspension, biomolecule separation,protein fractionation, cell separation, waste water treatment, waterpurification, bioreactors, and food processing.

For non-medical applications, the compositions described herein resistpreferably greater than 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, 99%, or 99.9% adsorption of a fouling material relative to anuncoated control for 1 day, 7 days, 14, 21, 30, 45, 60, 90, 120, 180,365, or 1000 days.

These materials can also be used to treat surfaces of fibers,particulates and films for the applications of textiles, additives,electric/optical appliances, packaging materials and colorants/inks.

The compositions described herein are stable over extended periods oftime, retaining at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, or 99% of their non-fouling,anti-thrombotic, and/or antimicrobial properties for extended periods oftime, for example, at least 1, 7, 14, 21, 30, 45, 60, 90, 120, 180, 365,or 1000 days.

EXAMPLES Example 1 Preparation of a Coated Substrate

Preparation of an Undercoating

An undercoating, with reactive functional groups for crosslinking, wasprepared through free radical polymerization. 2-aminoethyl methacrylate(AEMA, 20-80 mol %), lauryl methacrylate (LMA, 0-50 mol %),2-hydroxypropyl methacrylate (HPMA, 0-50 mol %) and3-(trimethoxysilyl)propyl methacrylate (TMOSMA, 0-25 mol %) were mixedin methanol. Azobisisobutyronitrile (AIBN), an initiator, was added andthe reaction solution was heated to 60-65° C. The reaction was allowedto proceed for 12-24 hours at 60-65 C, with gentle stirring, undernitrogen. The crude polymer was purified by dialysis against methanol.

Preparation of a Top Coating

A top coating, containing reactive functional groups for crosslinking,was prepared through free radical polymerization. Sulfobetainemethacrylate (SBMA, 25-70 mol %), glycidyl methacrylate methacrylate(GMA, 10-50 mol %), and 2-hydroxypropyl methacrylate (HPMA, 0-50 mol %)were mixed in methanol. Azobisisobutyronitrile (AIBN), an initiator, wasadded to the reaction solution, which was heated to 60-65° C. Thereaction was allowed to proceed for 24 hours at 60-65° C., with gentlestirring, under nitrogen. The crude polymer was purified by dialysisagainst deionized H₂O.

Coating a Substrate Surface with an Undercoating and Top Coating

The purified undercoating polymer was dissolved in methanol. A substratewas dipped into a solution of methanol containing the undercoatingpolymer. The thickness of the undercoating can be tailored by varyingthe concentration of the polymer in solution or by increasing the numberand speed of each dip step. After this first dipping treatment thecoating is cured in an oven (37-80° C.) for 10-24 hours. This heattreatment activates a crosslinking reaction between individual polymerchains, forming covalent bonds between polymer chains stabilizing theundercoating on the substrate surface. The resulting polymer-treatedsubstrate is then dipped in an aqueous solution containing purified topcoating polymer. The coating was cured in an oven at 37-80° C. for 12-48hours. After curing, the top coating polymer is covalently bound on theundercoating and the substrate is coated with a complete non-foulingcoating set.

Example 2 Synthesis of an Undercoating

A nitrogen purged solution of 3.4 g laurylmethacrylate, 4.25 ghydroxypropylmethacrylate, 1.56 g trimethoxysilylpropylmethacrylate,1.20 g aminoethylmethacrylamide hydrochloride salt, and 0.40 gazoisobutyronitrile in 90 mL anhydrous methanol was heated (64° C.) 14h. After allowing the mixture to cool to room temperature, the solutionwas dialyzed against anhydrous methanol using benzylated dialysis tubingto afford a solution of undercoating in methanol.

Example 3 Synthesis of a Top Coat

A nitrogen purged solution of 1.08 g glycidylmethacrylate, 4.2 gN-(3-sulfopropyl)-N-methacryloxyethyl-N,N-dimethylammonium betaine, 0.24g azoisobutyronitrile in 90 mL anhydrous methanol was heated (64° C.) 14h. After allowing the solution to cool to room temperature, the solutionwas decanted from the precipitate. The precipitate was washed twice withanhydrous methanol and dried under vacuum. This topcoat is referred toas the H topcoat.

Example 4 Top Coating Synthesis by Controlled Radical Polymerization

Copper (1) bromide was added to a flask (125 mL) containing a stir bar.The flask was sealed with a septum and the flask was flushed with argonfor 30 minutes Inhibitor was removed from glycidyl methacrylate (GMA)using an inhibitor removal column. Inhibitor free GMA (0.7 g) wasdiluted with 6.3 mL of methanol. This solution was placed in a separateflask, which was sealed with a septum. 3-Bromo-2-butanone (0.35 g) andmethanol (0.90 mL) were added to a separate flask. Bipyridine (0.4 g)was dissolved in methanol (8 mL), in a separate flask, which was sealedwith a septum. A 10% solution ofN-(3-sulfopropyl)-N-methacryloxyethyl-N,N-dimethylammonium betaine(SBMA) was prepared in 25 mL of a 75:25 mixture of methanol: water.Argon was bubbled through all of the solutions for 1 hour.

After one hour, bipyridine (1 mL) was added, by syringe, to the flaskcontaining the copper (I) bromide, followed by the addition of the GMAsolution (1.75 mL). To initiate the reaction, 3-bromo-2-butanone (0.046mL) was added, by syringe, to the reaction mixture containing the copper(I) bromide, GMA, and bipyridine. This reaction was allowed to proceedfor 1 hour at room temperature, with vigorous stirring. Following theinitial reaction, SBMA (45 mL) was added to the reaction flask. Thisreaction was allowed to proceed for 8 hours, before the reaction flaskwas opened to the air. Any remaining solution was decanted from theprecipitate that had formed. The precipitate was then dissolved inwater, centrifuged, and the solution was decanted and reserved. Thereserved solution was dried by lyophilization.

In another example both the GMA and SBMA monomers were added to thereaction flask at the same time.

Example 5 Coating Polyurethane Rods

Step 1. Preparing test articles. CARBOTHANE® (20% barium sulfate) rodextrusion (OD 0.118±0.002) was cut into 15 cm long test articles. Thetest articles were soaked in heptane for 5 minutes and then dried onaluminum foil for 1 hour at room temperature (RT).

Step 2. Dipping process in undercoating. The undercoating made inExample 2 was put into a cylindrical glass and held with clamps below anactuator that allowed the test articles to be lowered into and liftedout of the undercoating solution. The test articles were hung from theactuator with alligator clamps and lowered at a rate of ˜0.013 m/s. Oncethe test articles were completely immersed in the undercoating solution(5%) they were immediate lifted out of the solution at a rate of 0.012m/s. Once dipped, all test articles were hung with alligator clamps froma rack to dry at room temperature for 30 minutes.

Step 3. Undercoating curing process. After drying, all test articleswere transferred and hung in an oven for 16 hours at 60° C. to dry. Thetest articles were then removed from the oven and cooled to roomtemperature before dipping in the H-topcoat solution.

Step 4. Dipping process in H-topcoat. A solution (5%) of top coat madein Example 3 was prepared. The H-topcoat solution was put into acylindrical glass and held with clamps below an actuator that allowedthe test articles to be lowered into and lifted out of the H-topcoatsolution. The test articles were hung from the actuator with alligatorclamps and lowered at a rate of ˜0.013 m/s. Once the test articles werecompletely immersed in the topcoat solution they were immediate liftedout of the solution at a rate of 0.012 m/s. Once dipped, all testarticles were hung with alligator clamps from a rack to dry at roomtemperature for 30 minutes.

Step 3. H-topcoat curing process. After drying, all test articles weretransferred and hung in an oven for 40 hours at 60° C. The test articleswere then removed from the oven and cooled to room temperature (RT).

Example 6 Antimicrobial Activity

A model for biofilm formation of S. epidermidis ATCC 35984 using a CDCbiofilm reactor system has been developed. Briefly, the system consistsof a stirred glass reactor vessel with inlet and outlet ports forsterile media addition and removal, as well as a sample holder for ourmaterials. The culture conditions (media formulation and media flowrate) were modified slightly from those described in the current ASTMmethod (ASTM E2562-07) to allow robust surface growth of S. epidermidison our control PU materials, while minimizing planktonic bacterialgrowth. Materials were seeded with bacteria for 2 hrs (planktonicconcentration ˜1×10⁶ CFU/ml in PBS with agitation at 37° C.), placed inthe reactor system, and incubated for 24 hrs at 37° C. with a continualexchange of sterile media. After 24 hrs, accumulated biofilm onmaterials were removed by sonication and enumerated on TSA (tryptic soyagar) plates. Undercoat-topcoat formulations described in Example 5above have achieved 99% reduction in colonization in this assay relativeto uncoated polyurethane.

Example 7 Antimicrobial Activity after Serum Exposure

Antimicrobial activity of the samples produced in Example 5 was measureusing by using a colonization assay pre-incubation with 50% fetal bovineserum for 18-20 hours at 120 RPM at 37° C., which is preferred.Following pre-incubation, samples are placed in Staphylococcus aureus(S. aureus, ATCC 25923) which has been diluted from an overnight cultureto a planktonic concentration of 1−3×10⁵ CFU/mL in 1% tryptone soy broth(TSB). Samples are incubated with bacteria for 24-26 hrs with agitation(120 rpm) at 37° C. The concentration of TSB varies with the organismbeing used. After incubation, the samples are placed in 3 ml PBS for 5min at 240 RPM at 37° C. to remove bacteria not tightly attached. Thenaccumulated bacteria on materials are removed by sonication in a newsolution of PBS and the total number of bacterial cells quantifiedthrough dilution plating. A log reduction of 1.16 relative to uncoatedCARBOTHANE® was achieved.

We claim:
 1. A composition comprising a functionalized substrate havinga polymeric undercoat immobilized thereon and a zwitterionic polymerictopcoat copolymer covalently bound to the polymeric undercoat, thepolymeric undercoat being between the functionalized substrate and thezwitterionic topcoat copolymer, the polymeric undercoat and thezwitterionic polymeric topcoat copolymer comprising complementaryreactive functional groups wherein the zwitterionic polymeric topcoatcopolymer is covalently bound to the polymeric undercoat via covalentbonds formed by the complementary reactive functional groups, andwherein more than 30 mole % of the polymerized monomers comprised by thezwitterionic polymeric topcoat copolymer have the formula

wherein B is selected from the group consisting of:

R is selected from the group consisting of hydrogen, substituted alkyl,and unsubstituted alkyl; E is selected from the group consisting ofsubstituted alkyl, unsubstituted alkyl, —(CH₂)_(y)C(O)O—, and—(CH₂)_(y)C(O)NR²; R² is selected from hydrogen and substituted orunsubstituted alkyl; Y is an integer from 0-12; L is absent or is astraight or branched alkyl group optionally including one or more oxygenatoms; ZI is a zwitterionic group; and X is an integer from 3 to 1000.2. The composition of claim 1, wherein the substrate is selected fromthe group consisting of metallic materials, ceramics, polymers, wovenmaterials, non-woven materials, silicon, and combinations thereof. 3.The composition of claim 2, wherein the substrate is a metallic materialand the metallic material is selected from the group consisting oftitanium and alloys thereof, stainless steel, tantalum, palladium,zirconium, niobium, molybdenum, nickel-chrome, cobalt or alloys thereof,and combinations thereof.
 4. The composition of claim 2, wherein thesubstrate is a ceramic and the ceramic is selected from the groupconsisting of oxides, carbides, or nitrides of the transition metalelements or metalloid elements.
 5. The composition of claim 2, whereinthe substrate comprises a polymer and the polymer is selected from thegroup consisting of polystyrene and substituted polystyrenes,poly(urethane)s, polyacrylates and polymethacrylates, polyacrylamidesand polymethacrylamides, polyesters, polysiloxanes, polyethers,poly(orthoester), poly(carbonates), poly(hydroxyalkanoate)s,polyfluorocarbons, polyether ether ketone, silicones, epoxy resins,polyamides and copolymers thereof, nylon, polyalkenes, phenolic resins,polytetrafluoroethylene, natural and synthetic elastomers, adhesives andsealants, polyolefins, polysulfones, polyacrylonitrile, polysaccharides,and combinations thereof.
 6. The composition of claim 5, wherein thepolymer is polyurethane or polycarbonate-based polyurethanes.
 7. Thecomposition of claim 5 wherein the complementary reactive functionalgroups are epoxy-amine, isocyanate-carboxyl, glycidyl-anhydride,amine-anhydride, silanol-silanol, isocyanate-amine, carboxyl-amine, orhydroxyl-carboxyl.
 8. The composition of claim 2, wherein thecomplementary reactive functional groups are epoxy-amine,isocyanate-carboxyl, glycidyl-anhydride, amine-anhydride,silanol-silanol, isocyanate-amine, carboxyl-amine, or hydroxyl-carboxyl.9. The composition of claim 1, wherein the complementary reactivefunctional groups are epoxy-amine, isocyanate-carboxyl,glycidyl-anhydride, amine-anhydride, silanol-silanol, isocyanate-amine,carboxyl-amine, or hydroxyl-carboxyl.
 10. The composition of claim 9,wherein the undercoating comprises a homopolymer or copolymer formed bycondensation or radical polymerization of one or more monomers selectedfrom the group consisting of acrylates, acrylamides, vinyl compounds,di, tri-, and tetra-isocyanates, alcohols, amines, and thiocyanates;lactones, lactams, and combinations thereof.
 11. The composition ofclaim 10, wherein the undercoating comprises a homopolymer or copolymerformed by condensation or radical polymerization of one or more acrylatemonomers.
 12. The composition of claim 11, wherein the undercoatingcomprises a copolymer of glycidyl methacrylate (GMA), 2-hydroxypropylmethacrylate (HPMA), lauryl methacrylate (LMA), and trimethoxysilylmethacrylate (TMOSMA).
 13. The composition of claim 1, wherein thenon-fouling polymeric topcoat is a copolymer.
 14. The composition ofclaim 13, wherein greater than 35 mole % of the polymerized monomers ofthe copolymer comprise non-fouling moieties.
 15. The composition ofclaim 1, wherein ZI is selected from the group consisting of:

wherein R₃ and R₄ are independently selected from the group consistingof hydrogen and substituted or unsubstituted alkyl; R₅ is selected fromthe group consisting of substituted or unsubstituted alkyl, phenyl, andpolyether groups; and M is an integer from 1-7.
 16. The composition ofclaim 15, wherein the zwitterionic polymeric topcoat copolymer furthercomprises one or more polymerized monomers selected from the groupconsisting of acrylates, acrylamides, vinyl compounds, di, tri-, andtetra-isocyanates, alcohols, amines, and thiocyanates; lactones,lactams, and combinations thereof.
 17. The composition of claim 16,wherein the zwitterionic polymeric topcoat copolymer comprises 25-70 mol% sulfobetaine methacrylate, 10-50 mol % glycidyl methacrylatemethacrylate, and 0-50 mol % 2-hydroxypropyl methacrylate.